Gap junction proteins (connexins) form intercellular channels that permit the organized spread of activating current necessary for coordinated contractile function. Turnover of connexin43 (Cx43), the major ventricular coupling protein, is extraordinarily dynamic (t1/2 less than 2 hr). hr). Rapid changes in coupling occur under pathophysiological conditions and contribute to development of anatomic substrates of reentrant arrhythmias. Phosphorylation of Cx43 appears to play a critical role in channel assembly, conductance properties, and turnover dynamics. However, the specific phosphorylated residues and their functional and regulatory significance are unknown. The proposed studies will elucidate the biological role of Cx43 phosphorylation and test the hypothesis that dephosphorylation of specific phosphoamino acids targets Cx43 to specific proteolytic pathways. First, tandem mass spectrometry will be used to fully characterize the diversity of phosphorylation isoforms of Cx43 in normal ventricular myocytes Then, Cx43 dephosphorylation or proteolysis will be altered by pharmacological interventions in ventricular myocytes and changes in Cx43 phosphorylation patterns as well as changes in gap junction structure and function and Cx43 turnover dynamics will be measured to identify putative regulatory phosphorylation sites. Finally, site-directed mutagenesis and cell transfection strategies in communication-deficient cells will be used to formally test hypotheses about the regulatory and functional roles of specific phosphoamino acids in Cx43. Insights gained from these studies will contribute to a more mechanistic understanding of fundamental processes regulating intercellular coupling in the normal and diseased heart.